1. Field
The present application relates to a structured illuminating microscopy and a structured illuminating observation method.
2. Description of the Related Art
A super-resolution microscopy is one that modulates an illumination light flux that illuminates a sample plane and demodulates an image-forming light flux which is incident on a position that is substantially conjugated with the sample plane of an image-forming optical system, in order to make information regarding a high spatial frequency that exceeds a resolution limit (diffracted light at a large angle) out of diffracted light that exits from a sample, to be contributed to an image formation (refer to Non-Patent Document 1: W. Lukosz, “Optical systems with resolving powers exceeding the classical limit. II”, Journal of the Optical Society of America, Vol. 57, PP. 932-941, 1967, Non-Patent Document 2: Mats G. L. Gustafsson et al., “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination”, Proceedings of the SPIE—The International Society for Optical Engineering, Vol. 3919, pp. 141-150, 2000, Patent Document 1: U.S. Pat. No. 6,239,909, Patent Document 2: Specification of U.S. Pat. No. RE 38307, Patent Document 3: U.S. Pat. No. 8,115,806, and the like).
In a method of Non-Patent Document 1, a diffraction grating (diffraction grating for modulation) is disposed in the vicinity of a sample plane, and a diffraction grating (diffraction grating for demodulation) having a grating constant which is conjugated with that of the diffraction grating for modulation, is disposed at a position which is substantially conjugated with the sample plane of an image-forming optical system. When those two diffraction gratings are moved in a conjugated manner, it becomes possible to observe a structure of sample by separating it from patterns of the diffraction gratings.
Meanwhile, Patent Document 1 discloses an example in which a structured illuminating microscopy is applied to a fluorescent observation. In a method of Patent Document 1, a light flux that exits from a coherent light source is split into two light fluxes by a diffraction grating, and those two light fluxes are individually condensed on mutually different positions on a pupil of an objective lens. At this time, the two light fluxes exit from the objective lens as collimated light fluxes with different angles, and overlap each other on a sample plane to form striped interference fringes. Accordingly, the sample plane is subjected to structured illumination. Further, in the method of Patent Document 1, images of sample images are repeatedly obtained while shifting a phase of the structured illumination in steps, and calculation corresponding to the aforementioned separation (separating calculation) and calculation corresponding to the aforementioned demodulation (demodulating calculation) are performed on the obtained plurality of images.
Note that as a method of shifting the phase of structured illumination in steps, there are a method in which a wedge-shaped prism is inserted into one of the above-described two light fluxes and moved in steps in a direction perpendicular to an optical axis, a method in which a diffraction grating is moved in steps in a direction perpendicular to a grid line, a method in which a sample is moved in steps in a pitch direction of structured illumination, and the like.
However, when an optical element is moved in steps, a certain period of time is required for stopping the moving optical element at an appropriate position, so that it is difficult, in the method of Patent Document 2, to reduce a period of time taken for obtaining all of the required images. Particularly, when a sample being an observational object is an organism specimen, there is a chance that a structure of the sample changes every second, so that the obtainment of images should be performed as fast as possible.
Further, as an application of technique utilizing the interference fringes (Patent Document 1), a technique of turning a beam that contributes to the interference fringe into three beams (Non-Patent Document 2) has also been proposed for achieving a super-resolution effect in both of an in-plane direction and a depth direction of a sample. This is because, if three beams are used, a stripe pattern of structured illumination can be generated not only in the in-plane direction but also in the depth direction. However, in that case, the number of images required for the aforementioned separating calculation is increased, so that it can be considered that the necessity of increasing the speed of obtaining images is particularly high.